1. Field of the Invention
The present invention relates to an apparatus and method of assay in utilizing attenuated total reflection. More particularly, the present invention relates to an apparatus and method of assay in utilizing attenuated total reflection, in which density of analyte fluid can be adjusted with high precision, and thus analysis of interaction between ligand and the analyte can be easy with a high throughput by raising precision in retrieval of a reaction curve.
2. Description Related to the Prior Art
An assay apparatus in utilizing attenuated total reflection for assaying a sample is known in the field of the biosensor. U.S. Pat. No. 5,313,264 (corresponding to JP-A 4-501462) discloses a surface plasmon resonance (SPR) sensor as a typical example for this assay.
A thin film, or metal film, is formed on a transparent dielectric medium. One surface of the metal film is a sensing surface where reaction of a sample occurs. Another surface of the metal film is a metal/dielectric interface where light is applied by satisfying a condition of total reflection. The reaction is detected to assay the sample according to attenuation of the reflected light from the metal/dielectric interface. The surface plasmon resonance (SPR) assay apparatus is constructed to detect surface plasmon resonance created on the sensing surface which is the first surface of the metal film.
Surface plasmon is a term to mean the compressional wave created on the surface of the metal and included in plasmon as quantized expression of the compressional wave. In a metal, free electrons vibrate to generate the compressional wave called a plasma wave. The surface plasmon travels along the surface of the metal.
Light for detection is applied to a metal/dielectric interface of the metal film that is back to the sensing surface so that the total reflection condition is satisfied, namely at an angle of incidence equal to or more than a critical angle. In addition to the total reflection created on the metal/dielectric interface, a small component of the light passes through the metal film without reflection, and penetrates to the sensing surface. A wave of the penetrating component is called an evanescent wave. Surface plasmon resonance (SPR) is created when frequency of the evanescent wave coincides with that of the surface plasmon. In response to this, intensity of the reflected light attenuates remarkably. In the assay apparatus, the attenuation in the reflected light reflected by the metal/dielectric interface is detected, to recognize creation of the SPR on the sensing surface.
The angle of incidence, namely resonance angle of the light to generate the SPR depends on the refraction index of the transmission medium transmitting evanescent wave and surface plasmon. In other words, a change in the resonance angle to create SPR changes in response to a change in the refraction index of the transmission medium. The substance contacting the sensing surface is a transmission medium transmitting the evanescent wave and surface plasmon. If binding or dissociation between two molecules occurs on the sensing surface, the resonance angle changes because of a change in the refraction index of the transmission medium. In the SPR system, the change in the refraction index is detected, to measure interaction of molecules.
The assay apparatus can be used for various kinds of studies in a biochemical field or the like, for example to study interaction of protein, DNA and various biomaterials, and to select candidate drugs by screening. Also, the technique is useful in the fields of the clinical medicine, food industries and the like. It is possible to use one of two substances as a ligand and another of them as an analyte if those have bioaffinity. For the purpose of screening, protein as biomaterial is used as ligand. Candidate drugs are discretely used as analyte, and contacted with the ligand on the sensing surface, to study interaction.
JP-A 6-167443 and U.S. Pat. No. 5,822,073 disclose discloses an SPR assay apparatus in which an optical system of Kretschmann configuration is used for incidence of light to the metal film. According to the Kretschmann configuration, the surface of the metal film as metal/dielectric interface is fitted on a prism, which condenses light and directs the light to the metal/dielectric interface in a manner conditioned for total reflection. A sample or ligand is immobilized on the sensing surface. A flow channel is formed to have the sensing surface inside, and causes analyte fluid to flow. The analyte fluid is introduced in the flow channel to flow, and is caused to contact the ligand. Interaction between the analyte fluid and the ligand is assayed by detecting surface plasmon resonance created during the reaction.
In the surface plasmon resonance (SPR) assay, interaction between the ligand and analyte on the sensing surface can be output rapidly as an output signal. A reaction speed constant is obtained by use of a reaction curve which represents changes of the output signal with time. The interaction between the ligand and analyte can be analyzed with the reaction speed constant in a time sequential manner. To obtain the reaction speed constant, the reaction curve according to the measurement is subjected to curve fitting based on the calculation, for example non-linear least squares fitting.
Forms of the reaction curve suitable for the curve fitting are known according to various experimental attempts. For the purpose of obtaining an optimized form of the reaction curve, density of analyte solution or analyte liquid is changed in a stepwise manner, to measure an output signal of the surface plasmon resonance (SPR).
If unknown compounds are treated for measurement, for example drug screening, possibility of reaction itself is not known initially. To this end, the analyte solution or analyte liquid of a high density is prepared to measure the compounds, to check occurrence or lack of reaction. If reaction occurs, the analyte liquid of a second density lower than the first density is prepared, to measure the compound for the second time. For example, an initial rise of the signal in an early section can be too high. In this situation, the analyte liquid of a third density lower than the second density is prepared, to measure the compound for the third time. According to repeated experiments, an optimized form of the reaction curve is obtained finally. After the successful result of the optimization, the curve fitting is made according to the reaction curve, to find the reaction speed constant.
Calculating processes of the curve fitting are known in the art. The reaction speed constant may be obtained readily by utilizing software of arithmetic processing only if the reaction curve can be optimized.
However, determination of density of the analyte solution or analyte liquid and preparation of the analyte liquid have been based on skills of technicians conducting experiments for the purpose of optimizing the reaction curve for the curve fitting. Errors in observation, estimation and calculation of the technicians are very likely to occur according to specifically personal skills. This leads to a long time analysis or an erroneous result of the analysis, which is very unsuitable for assay with high precision.